猴副流感病毒5型实时荧光定量PCR方法的建立与初步应用

目的建立猴副流感病毒5型（SV5）实时荧光定量PCR方法，并初步应用于猴样本检测。方法根据SV5病毒NP基因保守片段设计特异引物和TaqMan探针，使用分子生物学方法制备质粒标准品，建立SV5实时荧光定量PCR方法；对该方法的线性、敏感性、特异性及稳定性进行测定；并使用该方法对24个猴样品进行检测。结果成功建立SV5实时荧光定量PCR方法；该方法线性范围为（6．8×10^9～6．8×10^5）copy／μL，灵敏度为6．8copy／μL，批内变异系数（CV）为0．63％～8．71％，批间CV为1．52％-12．38％，而且方法特异性好；在24个猴肺样品中未检测出阳性。结论该方法的建立为实验动物和动物源性生物制品中SV5的定量检测奠定了基础。

Establishment of Real time RT-PCR Method for Detection of Simian Parainfluenza Virus (SV5) and Its Preliminary Application

Objective To establish a real-time RT-PCR method for detection of simian parainfluenza virus type 5 （SV5） in biological materials from monkeys and explore its application. Methods According to SV5 virus NP gene fragments, a pair of specific primers and TaqMan probe were designed and synthesized. SV5 RNA standards were prepared using molecular biological techniques. Then the linearity, sensitivity, specificity and stability of the method were determined. Last, this method was used for testing lung samples of 24 monkeys. Results A real-time PCR method for detection of SV5 was successfully established. The linear range was 6.8 × 10^9 copies/μL to 6.8 × 10^5 copies/μL, sensitivity was 6.8 copies/μL, coefficient of variation （CV） was 0.63% to 8.71% , inter-assay CV was 1.52% to 12. 38% , and the method was highly specific. No positive reaction was detected in the lung samples of 24 monkeys. Conclusions The establishment of this method provides a useful basis for quantitative detection of SV5 in laboratory animals and animal-derived biological products.